To study the possibility of cross-species transmission of CWD, two species of nonhuman primates, squirrel monkeys and cynomologus macaques, were infected orally or intracerebrally with brain material derived from CWD affected deer or elk. At the conclusion of FY13 thirteen of thirteen intracerebrally infected squirrel monkeys had developed clinical neurological signs and were confirmed by biochemical and pathological testing of brain to have a prion disease. At the conclusion of FY14 11 of 11 orally infected squirrel monkeys also developed disease. At the conclusion of FY16 a total of twelve (2 in FY16)CWD-infected cynomologus macaques had been euthanized and screened for prion disease. None of these twelve cynomologus macaques have developed prion disease regardless of inoculation route. Three cynomologus macaques remain on this portion of the study and plans to euthanize and screen the remaining monkeys for prion disease at the end of FY16 are in place. During FY08 two cynomologus macaques and two squirrel monkeys were inoculated with brain homogenates derived from a CWD infected squirrel monkey to evaluate the ability of CWD to adapt to a nonhuman primate host. During FY10 both squirrel monkeys inoculated with squirrel monkey adapted CWD developed disease. The incubation period from this inocula was much faster and clinical signs were slightly different suggesting adaptation to a new host or a higher titer of inocula. The successful passage of CWD derived from a squirrel monkey into additional squirrel monkeys confirms the presence of prion infectivity. This confirmation was important since the same squirrel monkey CWD inocula did not cause disease in transgenic mice expressing human or deer prion protein and has not caused disease in any of the cynomolgus macaques still under study. During FY13 one of the cynomologus macaques was euthanized due to chronic weight loss and liver failure. This monkey was tested for TSE disease and was negative, indicating that squirrel monkey adapted CWD may still be unable to infect other species on non-human primates. The remaining monkey in this study will be euthanized and tested for prion disease at the end of FY16. In addition to testing infected squirrel monkey brain we are also evaluating blood from infected squirrel monkeys for the presence of prion infectivity. None of the blood product recipient monkeys have shown any signs of disease at the close of FY16. To further test adaptation of CWD to either a human tropism we inoculated material from the clinically ill second passage squirrel monkeys into transgenic mice expressing human prion protein (tg66) during FY11. During FY12 these tg66 mice were euthanized and tested for prion disease. All of the mice in this second passage study were negative for prion infection. The lack of transmission to the humanized mice is encouraging data that supports the conclusion that CWD will likely not infect humans. During FY15 we started a study to directly test the transmission of cervid dervied CWD into the tg66 mice. These mice have been inoculated and have not developed disease at the end of FY16. They will continue to be monitored for prion disease onset.